Bacterial toxins are the causative agents for a variety of human diseases. However, the molecular basis of infection, in many cases, remains enigmatic. Cholera toxin (CT) produced by Vibrio cholerae is the virulence factor responsible for massive secretory diarrhea. As this disease remains a global health issue, elucidating its basic mechanism of action is paramount. To intoxicate cells, CT is transported from the cell surface to the lumen of the endoplasmic reticulum (ER). In this compartment, the toxic CTA1 fragment of CT disguises as a misfolded protein and hijacks the cellular machinery that normally moves misfolded proteins from the ER into the cytosol for degradation by the proteasome. Upon reaching the cytosol, CTA1 however escapes proteasomal destruction and triggers a signaling cascade that leads to pathologic water secretion (i.e. diarrhea), which can lead to death in severe cases. How CTA1 is transferred from the ER into the cytosol, a decisive intoxication step, remains poorly understood. In this application, we intend to address this question by using a combination of biochemical and cell biological approaches. Historically, studies on pathogen-host cell interactions have expounded on basic cellular processes. Moreover, these findings often led to the identification of key molecular targets amenable for therapeutic intervention. Thus we anticipate that our findings are likely to reveal novel mechanisms of protein transport across biological membranes and to identify new cellular factors that may serve as viable therapeutic targets. In addition, as other toxins such as ricin and shiga toxin also undergo ER-to-cytosol transport to induce cytotoxicity, our results should provide insights into their mechanism of action as well. PUBLIC HEALTH RELEVANCE: Cholera toxin (CT) causes pathologic water secretion (i.e. diarrhea) in animals, which can lead to death in severe cases. A decisive step in the toxin-dependent infection process is transport of the toxin across the membrane of a sub-cellular compartment known as the endoplasmic reticulum (ER). However, the molecular details by which CT penetrate the ER membrane is not clear. We intend to clarify these processes in this application.